mitochondrialcholesterolcontributestochemotherapy ...because strategies targeting mitochondria have...

Mitochondrial Cholesterol Contributes to Chemotherapy

Resistance in Hepatocellular Carcinoma

Joan Montero,1,2

Albert Morales,1,2

Laura Llacuna,1,2

Josep M. Lluis,1,2

Oihana Terrones,3

Gorka Basanez,3Bruno Antonsson,

4Jesus Prieto,

2,5Carmen Garcıa-Ruiz,

1,2

Anna Colell,1,2

and Jose C. Fernandez-Checa1,2

1Liver Unit and Centro de Investigaciones Biomedicas Esther Koplowitz, IMDiM, Hospital Clınic i Provincial, Institut d’InvestigacionsBiomediques August Pi i Sunyer, and Department of Cell Death and Proliferation, Instituto de Investigaciones Biomedicas deBarcelona, Consejo Superior de Investigaciones Cientıficas, Barcelona, Spain; 2Centro de Investigacion Biomedica en Red(CIBERehd); 3Unidad de Biofısica (Centro Mixto Consejo Superior de Investigaciones Cientıficas-Universidad del PaısVasco/Euskal Herriko Unibertsitatea), Universidad del Paıs Vasco/Euskal Herriko Unibertsitatea, Bilbao, Spain; 4MerckSerono Internacional, Geneva, Switzerland; and 5Liver Unit and Division of Hepatology and Gene Therapy,University Clinic and Center for Applied Medical Research, University of Navarra, Pamplona, Spain

Abstract

Cholesterol metabolism is deregulated in carcinogenesis, andcancer cells exhibit enhanced mitochondrial cholesterolcontent whose role in cell death susceptibility and cancertherapy has not been investigated. Here, we describe thatmitochondria from rat or human hepatocellular carcinoma(HC) cells (HCC) or primary tumors from patients with HCexhibit increased mitochondrial cholesterol levels. HCCsensitivity to chemotherapy acting via mitochondria isenhanced upon cholesterol depletion by inhibition of hydrox-ymethylglutaryl-CoA reductase or squalene synthase (SS),which catalyzes the first committed step in cholesterolbiosynthesis. HCC transfection with siRNA targeting thesteroidogenic acute regulatory protein StAR, a mitochondrialcholesterol–transporting polypeptide which is overexpressedin HCC compared with rat and human liver, sensitized HCCto chemotherapy. Isolated mitochondria from HCC withincreased cholesterol levels were resistant to mitochondrialmembrane permeabilization and release of cytochrome c orSmac/DIABLO in response to various stimuli including activeBax. Similar behavior was observed in cholesterol-enrichedmitochondria or liposomes and reversed by restoring mito-chondrial membrane order or cholesterol extraction. More-over, atorvastatin or the SS inhibitor YM-53601 potentiateddoxorubicin-mediated HCC growth arrest and cell deathin vivo . Thus, mitochondrial cholesterol contributes to chemo-therapy resistance by increasing membrane order, emergingas a novel therapeutic niche in cancer therapy. [Cancer Res2008;68(13):5246–56]

Introduction

Cholesterol is an integral component of cellular membranes thatplays an essential role in maintaining their integrity and function(1). In addition to the regulation of membrane order, cholesterolinduces membrane packing in lateral microdomains (rafts) of theplasma membrane, providing a scaffold for a variety of membrane-

associated signaling proteins (2, 3). Due to this role in modulatingmembrane structure and function, cholesterol levels in cellmembranes are tightly regulated. The main sources of cellularcholesterol involve either its uptake from cholesterol-rich low-density lipoproteins or its de novo synthesis through theconversion of 3-hydroxy-3-methylglutaryl-CoA into mevalonate by3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR), the rate-limiting step in cholesterol synthesis, which is transcriptionallyregulated by endoplasmic reticulum–based transcription factorSREBP-2 (1, 4).

Cholesterol accumulation and enhanced cholesterol-rich lipidrafts have been reported in several solid tumors that modulatetumor cell growth and survival by activating particular signalingpathways such as Akt (5, 6). In addition, cholesterol metabolism isabnormal in many malignancies with loss of cholesterol feedbackand HMG-CoAR up-regulation despite enhanced cholesterol levels(7). Moreover, malignant cells exhibit elevated levels of mevalonate,which has been shown to promote tumor growth in vivo and pro-liferation of breast cancer cells (8). Consistent with this scenario,statins, which block HMG-CoAR by competing with mevalonatefor binding to the active site, have been proposed for cancerprevention and/or treatment, although their efficacy as anticanceragents has not always been established (9). Furthermore, evenin those cases where statins showed promising results, it re-mained unclear whether the therapeutic effects were due to thecholesterol-lowering activity or to the down-regulation of iso-prenoids, which are known to modulate multiple proteins byposttranslational modifications (9, 10).

Mitochondria are cholesterol-poor organelles with estimatesranging from 0.5% to 3% of the content found in plasmamembranes (1, 11). However, unphysiologic mitochondrial choles-terol levels have been described in solid tumors. For instance,mitochondrial cholesterol levels of tumors from Buffalo ratsbearing transplanted Morris hepatomas were 2- to 5-fold higherthan the content found in mitochondria prepared from host liver,and correlated with the degree of tumor growth and malignancy(12–15). Although the mechanisms underlying the mitochondrialcholesterol accumulation in cancer cells are poorly understood,recent observations have reported the activity of cholesterol-transporting polypeptides, including the steroidogenic acuteregulatory protein (StAR) in human HepG2 cells that contributeto the mitochondrial intermembrane trafficking of cholesterol (16).Although cholesterol enrichment in mitochondria can impairspecific mitochondrial components accounting, in part, for themitochondrial dysfunction described in cancer cells (13, 15, 17–20),

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

A. Colell and J.C. Fernandez-Checa share senior authorship.Requests for reprints: Jose C. Fernandez-Checa, Liver Unit, Hospital Clınic i

Provincial, C/Villarroel, 170, 08036-Barcelona, Spain. Phone: 34-93-227-5709; Fax: 34-93-451-5272; E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-07-6161

Cancer Res 2008; 68: (13). July 1, 2008 5246 www.aacrjournals.org

Research Article

Research. on August 29, 2020. © 2008 American Association for Cancercancerres.aacrjournals.org Downloaded from

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its effect in cell death susceptibility and cancer therapy has notbeen previously examined. This may be of potential relevancebecause strategies targeting mitochondria have been proposedas potential use in cancer therapy (21), and alterations in themitochondrial apoptotic pathway, particularly the modulationof the mitochondrial membrane permeabilization (MMP), maycontribute to cancer growth and desensitization to cancer therapy(21, 22).

Hepatocellular carcinoma (HC) is one of the main causes ofcancer-related deaths that frequently arises on a background ofchronic inflammation and exhibits high resistance to currenttherapy (23). Thus, because mitochondria are known to play a keyrole in cell death (22) and cholesterol-regulated mitochondrialmembrane order has been reported to modulate MMP and thesubsequent release of apoptotic proteins (19), the purpose ofthis study was to examine the role of mitochondrial cholesterol inthe susceptibility of HC cells (HCC) to chemotherapy in vitro , themechanisms involved, and the relevance in an in vivo HC model.Our findings indicate that mitochondrial cholesterol contributes tochemotherapy resistance through altered membrane order, henceemerging as a novel therapeutic target in cancer therapy.

Materials and Methods

Materials and recombinant proteins. Dioleoylphosphatidylcholine,

tetraoleoylcardiolipin, and cholesterol were purchased from Avanti Polar

Lipids. Dodecyl octaethylene glycol monoether (C12E8), Methyl-h-cyclodex-

trin, melittin, Staphylococcus aureus a-toxin, tetanolysin, and fluoresceini-sothiocyanate-labeled dextrans of 70 kDa (FD-70) were obtained from

Sigma. Recombinant full-length human Bax with an amino-terminal His6

tag (Bax), caspase 8–cleaved murine BID with an amino-terminal His6 tag(tBid), and human Bcl-2 lacking the carboxy-terminal hydrophobic domain

(Bcl-2DC) were purified as previously described (24). Oligomeric Bax (oligo-

Bax) was obtained by incubating Bax in 100 mmol/L KCl, 10 mmol/L

HEPES, 0.1 mmol/L EDTA (pH 7.0) buffer (KHE buffer) containingoctylglucoside (2%, w/v) for 1 h at 4jC.

Cell culture, hepatocyte isolation, and mitochondria and mitoplastspreparation. The human hepatoblastoma cell line, HepG2, and the rat

hepatoma cell line, Reuber H35, were both obtained from the EuropeanCollection of Animal Cell Cultures and grown at 37jC in 5% CO2. Culture

medium was supplemented with 10% fetal bovine serum (FBS), 2 mmol/L

L-glutamine, and 1% nonessential amino acids and antibiotics. In someexperiments, 10% of delipidated FBS was used. Primary rat hepatocytes

were isolated by collagenase perfusion and cultured as described previously

(25). Cell viability in response to chemotherapy was determined by trypan

blue exclusion. Rat and human liver mitochondria were isolated asdescribed in Supplementary Methods. Mitochondria from HCC were

obtained by rapid centrifugation through Percoll density gradient as

described previously (19). Mitoplasts and outer mitochondrial membranes

were prepared by the fractionation of rat liver mitochondria with digitoninas described previously (19), monitoring the monoamine oxidase activity

for efficiency. In some experiments, the mitochondrial suspension

was incubated with 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylcyclopropyl)

octane (A2C, 125 nmol/mg protein) at 37jC for 30 min as described indetail (19).

Human hepatocarcinoma samples. Fresh-frozen samples from tumor

lesions were obtained from 6 patients (men, ages 57–73 y) with HC andapproved by the ethics committee. The samples were collected from the

surgical specimen after resection (n = 2) or from the liver explant at

transplantation (n = 4). In four cases, the tumor was multifocal, and in two

cases, uninodular. The underlying liver disease was alcoholic cirrhosis inthree cases, Hepatitis B virus–induced cirrhosis in two, and Hepatitis C

virus–induced cirrhosis in one. In addition, normal liver tissue was obtained

from the surgical specimen after partial hepatectomy because of colorectal

cancer metastatic to the liver.

Determination of cholesterol and phospholipid levels. The amountof cholesterol in mitochondria was measured by high performance liquid

chromatography (HPLC) using a Waters ABondapak C18 10-Am reversed-

phase column (30 cm � 4 mm inner diameter; ref. 26). The quantitation of

phospholipids is described in detail in the Supplementary Methods.Immunocytochemistry and laser confocal imaging. Cells were fixed

for 10 min with 3.7% paraformaldehyde in 0.1 mol/L phosphate buffer

before permeabilization with 0.1% saponin in 0.5% bovine serum albumin

(BSA)/PBS buffer for 5 min. Cells were incubated for 1 h with mousemonoclonal antibody anti–cytochrome c (1:200; PharMingen), rinsed with

PBS, and incubated for 45 min with the secondary antibody. In some cases,

filipin (50 Ag/mL) was added during the secondary antibody incubation as

described before (27). Images were obtained by confocal microscopy asdescribed in Supplementary Methods.

Modulation of cholesterol content and membrane order determi-nation. Cholesterol enrichment was achieved by incubating rat livermitochondria with a cholesterol-BSA complex at room temperature for

5 min as described (19). Parallel control experiments were performed using

only BSA. Cholesterol depletion in mitochondrial membranes from HepG2

or H35 cells was achieved by treatment with Me-h-cyclodextrin (MCD;40 mmol/L) for 30 min. Mitochondrial membrane order was evaluated by

fluorescence anisotropy of the mitochondria-bound dye 1,6-diphenyl-1, 3, 5-

hexatriene (DPH) as described previously (17, 19), determining the SDPH

from the steady-state fluorescence anisotropy values as described (28).Silencing of StAR by siRNA. The siRNA-targeting StAR and scrambled

siRNA were commercially purchased from Santa Cruz Biotechnology, Inc.

Transfection was performed using Lipofectamine2000 (Invitrogen) follow-ing the instruction of the manufacturer. Briefly, 5 � 105 HepG2 or H35 cells

were incubated with the transfection mixtures containing 100 pmol of the

siRNA-targeting StAR or the scrambled control siRNA. Cells were assayed

48 h after transfection for mRNA and protein StAR levels, and forsusceptibility to chemotherapy.

HCC xenograft model and treatment. Five- to six-week-old male

BALB/c athymic (nu/nu) nude mice were kept under pathogen-free

conditions and given free access to standard food and sterilized water.

All procedures were performed according to protocols approved by the

Institut d’Investigacions Biomediques August Pi i Sunyer Ethical Commit-

tee. HepG2 cells (2.5 � 106 in 200 AL of PBS) were injected s.c. into the

flanks of the mice. Tumors were measured periodically with a vernier

caliper, and the volume was calculated as length � width2 � 0.5, which has

been validated previously in comparison with other established methods

(29). After 2 to 3 wk, mice were randomly divided into 3 experimental

groups: group A, solvent (control); group B, atorvastatin (10 mg/kg); and

group C, YM-53601 (15 mg/kg). After 2 wk of daily treatment by p.o. gavage,

some animals received an i.p. injection of doxorubicin (10 mg/kg).

Differences in tumor volume during the next week were evaluated in each

group and expressed as percentage of change in tumor growth with respect

to the volume measured before chemotherapy administration.

Terminal deoxynucleotidyl-transferase–mediated dUTP nick-endlabeling assay and tumor progression. After sacrifice, tumors werefixed and paraffin sections (5 Am) from each area were stained with

terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling

(TUNEL) reagent using a commercial kit (In Situ Cell Death Detection kit;

POD from Roche). Immunohistochemical staining of CD34, a specificendothelial cell marker commonly used for microvessel quantification, was

performed with rat monoclonal anti-CD34 antibody (Abcam) at a dilution

of 1:50 (2 mg/mL). The slices were examined with a Zeiss Axioplan

microscope equipped with a Nikon DXM1200F digital camera. Seruma-feto protein levels were measured by the Centro Diagnostico Medico

(Hospital Clinic).

Statistics. Results were expressed as mean F SD with the number ofindividual experiments detailed in figure legends. Statistical significance

of the mean values was established by the two-tailed distribution Student’s

t test.

Supplementary methods. This section describes the procedures forWestern blot analysis, quantitative real-time PCR, large unilamellar vesicle

(LUV) preparation, and fluorimetric assays.

Mitochondrial Cholesterol and Hepatocellular Carcinoma

www.aacrjournals.org 5247 Cancer Res 2008; 68: (13). July 1, 2008



Results

Mitochondrial cholesterol in established HCC. Becauseprevious findings reported enhanced mitochondrial cholesterolcontent in solid Morris hepatoma tumors with respect to hostliver (12–15), we first assessed the mitochondrial cholesterol levelsfrom established HCC. HepG2 and H35 cells were fractionatedinto mitochondria and analyzed for lipid composition. Electronmicroscopy and Western blot analysis of PERK, Na+/K+ ATPase a1,and Rab5A indicated insignificant contamination with endoplas-mic reticulum, plasma membrane, and endosomes, respectively, inthe final mitochondrial fraction (Fig. 1A). Monitoring Lamp1 levelsby Western blot analysis further indicated the lack of lysosomalcontamination in the final mitochondrial fraction (Fig. 1A). Asshown, the total cholesterol levels in mitochondria from H35 andHepG2 cells were 3- to 10-fold higher with respect to rat andhuman liver mitochondria without changes in total phospholipidscontent (Fig. 1B), confirming previous results in implanted hepa-toma tumors in vivo (12–15). Interestingly, the levels of mitochon-drial cholesterol from primary tumors of patients with HC werehigher than the content of mitochondria from nontumor humanliver tissue and similar to those found in HepG2 cells (Fig. 1B).Moreover, the enhanced levels of free cholesterol of H35 or HepG2cells examined by confocal microscopy by filipin staining colo-calized with mitochondria, and this increase was further verified byHPLC analyses of isolated mitochondria (Fig. 1C). Finally, thesefindings on mitochondrial cholesterol up-regulation correlatedwith enhanced expression of the transcription factor SREBP-2 andHMG-CoAR in H35 and HepG2 cells with respect to rat and humanliver samples (Fig. 1D). Together, these findings validate the useof HCC lines to study the role of mitochondrial cholesterol inchemotherapy susceptibility.HMG-CoAR or squalene synthase inhibition sensitizes HCC

to mitochondria-targeted chemotherapy. We next examinedthe role of mitochondrial cholesterol in the susceptibility of HCCto chemotherapy targeting mitochondria. Arsenic trioxide orlonidamine, a derivative of indazole-3-carboxylic acid, are bothantineoplastic drugs that target mitochondria and induce mito-chondrial permeability transition (MPT; refs. 30, 31). Thapsigargin-based prodrugs developed for the treatment of prostate cancer(32) are potent inhibitors of endoplasmic reticulum Ca2+ ATPasesand induce MPT by Ca2+ overload, whereas doxorubicin, an anth-racycline antibiotic drug, stimulates mitochondrial reactive oxygenspecies (ROS) generation (33). Both cell lines displayed a reducedsusceptibility to increasing doses of thapsigargin, lonidamine,arsenic trioxide, or doxorubicin compared with primary rat hepa-tocytes (Fig. 2A). Various statins including lovastatin or atorvas-tatin have been shown to be effective in reducing cholesterol levelsin HepG2 cells (34). Lovastatin pretreatment sensitized H35 cellsto mitochondria-targeting drugs (Fig. 2B) with similar resultsobserved with HepG2 cells (Supplementary Fig. S1). Dying cellsdisplayed apoptotic features, such as caspase-3 activation (Supple-mentary Fig. S2A) and chromatin disruption (SupplementaryFig. S2B). Moreover, the susceptibility of H35 cells to bafilomycinA, an inhibitor of the vacuolar type H+-ATPase that impairslysosomal function and promotes apoptosis independently ofMMP (35), was not increased by lovastatin (Supplementary Fig. S1;Fig. 2B) and was not accompanied by enhanced cytochrome crelease (Supplementary Fig. S3), suggesting that the sensitizingeffect of lovastatin is specific for mitochondrial-targeting drugs.To further verify the specific role of cholesterol in chemotherapy

resistance, we investigated the effect of SS inhibition, which blockscholesterol biosynthesis without affecting the isoprenoid meta-bolism (Supplementary Fig. S4). Cell treatment with YM-53601, aspecific SS inhibitor (36), at a dose nontoxic to primary rat hepa-tocytes (Supplementary Fig. S5), potentiated the susceptibility ofH35 cells to thapsigargin, lonidamine, and doxorubicin (Fig. 2C)to a similar extent as seen by lovastatin treatment. Importantly,both lovastatin and YM-53601 reduced the mitochondrial choles-terol levels in both H35 and HepG2 cells (Fig. 2D). Finally, thesusceptibility of H35 cells to doxorubicin by lovastatin was prev-ented by mevalonate, independently of the inhibition of farnesyl-transferase, whereas mevalonate failed to restore resistance todoxorubicin after SS inhibition (Supplementary Fig. S6). Moreover,7-dehydrocholesterol, the immediate precursor of cholesterolsynthesis (Supplementary Fig. S4), decreased the susceptibilityto doxorubicin by SS inhibition (Supplementary Fig. S6). Collec-tively, these findings suggest the involvement of mitochondrialcholesterol in the resistance of HCC to drug-induced cell deathindependently of alterations in isoprenoid metabolism.siRNA-mediated StAR silencing sensitizes HCC to chemo-

therapy. To further substantiate that mitochondrial cholesterolenrichment in HCC contributes to mitochondrial-targeting che-motherapy resistance, we investigated the role of silencing StAR.StAR is a cholesterol-transporting polypeptide involved in theintramitochondrial trafficking of cholesterol, which regulates thesynthesis of steroids in specialized tissues (11, 16). The levels ofStAR were higher in H35 and HepG2 cells compared with rat andhuman liver samples being more abundant in HepG2 cells than inH35 cells (Fig. 3A), which correlated with the mitochondrialcholesterol levels observed in these cell lines (Fig. 1B). Transfectionwith siRNA-targeting StAR resulted in a significant reduction ofStAR protein and mRNA expression compared with cells tranfectedwith scrambled control siRNA (Fig. 3B), causing a significantreduction of mitochondrial cholesterol levels (Fig. 3C). Moreimportantly, the susceptibility of HepG2 cells to doxorubicin,thapsigargin, or arsenic trioxide was potentiated by transfectionwith StAR siRNA (Fig. 3D), with similar results observed with H35cells (Fig. 3D). Thus, these findings validate the observations withHMG-CoAR and SS inhibition, further supporting a key role formitochondrial cholesterol in the sensitization of HCC to chemo-therapy targeting mitochondria.Mitochondrial cholesterol modulates membrane order and

MPT. Cholesterol, particularly free cholesterol, regulates mem-brane physical properties (1, 27, 28). Thus, we next determined themembrane order from the steady-state fluorescence anisotropy ofDPH-labeled mitochondria. Mitochondria from H35 and HepG2cells showed higher membrane order (SDPH) compared withmitochondria from rat and human liver (Fig. 4A). Phospholipidscan also regulate membrane order. However, the content ofphosphatidylcholine, the major phospholipid in membranes,remained unchanged in mitochondria from H35 and HepG2 cellswith respect to rat or human liver samples, whereas phosphatidyl-ethanolamine levels in mitochondria from HepG2 cells but not H35cells were slightly lower than those found in human livermitochondria (Supplementary Fig. S7). Moreover, mitochondriafrom H35 and HepG2 cells exposed to the cholesterol-bindingagent MCD exhibited a significant depletion of cholesterol levelscompared with untreated mitochondria, which translated inreduced membrane order (Fig. 4A). In contrast, incubation of ratliver mitochondria with a cholesterol-albumin complex (19)resulted in significant cholesterol loading, reaching the levels

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Figure 1. Increased cholesterol content in mitochondria from rat (H35 ), human (HepG2 ) hepatoma cell lines, and human HC samples. A, electron microscopyanalysis showing purified mitochondria from H35 and HepG2 cells after cellular subfractionation (scale bars, 1 Am); and Western blot analysis of PERK, Na+/K+ATPasea1, Rab5A, Lamp1, and cytochrome c expression in homogenates (H ) or mitochondrial fraction (Mit ) from H35 cells. B, total cholesterol (black bars ) and phospholipidlevels (white bars ) performed by HPLC on lipid extracts from the mitochondrial fraction from H35, HepG2 cells, and human HC samples. C, colocalization ofmitochondria and free cholesterol by confocal microscopy using mouse anti–cytochrome c (cyt C ) and filipin, respectively. The graphs on the bottom representthe fluorescence intensity profiles plotted from a to b direction for the different cell lines. In addition, mitochondrial-free cholesterol content analyzed by HPLC.Results in B and C are mean F SD values from at least three independent experiments. * and **, P < 0.01 versus rat and human liver mitochondria, respectively.D, quantitative real-time reverse transcription-PCR mRNA expression of SREBP-2 and HMG-CoA reductase in H35 and HepG2 cells compared with rat and humanliver, respectively. Absolute mRNA values were determined, normalized to hypoxanthine phosphoribosyltransferase and reported as relative levels referred to theexpression in nontumor counterpart. Prot, protein.





found in mitochondria from H35 cells that increased themembrane order (Fig. 4A). Indeed, the mitochondrial membraneorder correlated with the cholesterol content (Fig. 4A). Cholesteroldistribution after cholesterol enrichment by the cholesterol-albumin complex was estimated in mitoplasts. Although the bulkof cholesterol (60–70%) was found in the outer membrane, inagreement with previous findings (19), the levels of cholesterol inmitoplasts from cholesterol-enriched mitochondria were higher(2- to 3-fold) than those found in mitoplasts from controlmitochondria (data not shown).

We next analyzed the role of cholesterol in the response ofmitochondria to MPT triggers. Isolated mitochondria from H35 cellswith or without MCD treatment were incubated with the superoxideanion-generating system, xanthine plus xanthine oxidase (X-XO),shown to induce the release of mitochondrial cytochrome c (37).Although rat liver mitochondria released cytochrome c in response

to X-XO, mitochondria from H35 were resistant to X-XO–inducedcytochrome c release (Fig. 4B); moreover, cholesterol extraction byMCD restored the sensitivity of mitochondria from H35 cells toX-XO–mediated cytochrome c release (Fig. 4B), with similar findingsobserved in mitochondria from HepG2 cells (Fig. 4B).

Ca2+ induces the transition of the MPT pore causingmitochondrial matrix swelling and release of the proapoptoticproteins (38). Rat liver mitochondria enriched in cholesterolby the cholesterol-albumin complex were resistant to swelling(data not shown), and cytochrome c and Smac/DIABLO release(Fig. 4C) induced by Ca2+. Moreover, mitochondria from H35 cellswere insensitive to Ca2+-induced Smac/DIABLO and cytochromec release (Fig. 4D), whereas cholesterol depletion by MCDrestored the response to Ca2+ (Fig. 4D). Thus, mitochondrial cho-lesterol regulates membrane order and the release of apoptoticproteins by MPT triggers.

Figure 2. Inhibition of cholesterolsynthesis sensitizes cells to differentcompounds that act on mitochondria.A, primary rat hepatocytes, H35, andHepG2 cells were incubated withincreasing doses of arsenic trioxide (ATO ),lonidamine, thapsigargin (Thg ), ordoxorubicin for 24 h. Cell viability wasdetermined by trypan blue exclusion. Atleast 100 cells in 4 different fields werecounted and expressed as a percentageof total cells. B and C, disruption ofcholesterol biosynthesis by lovastain orby the SS inhibitor YM-53601 (YM )sensitizes H35 cells to chemotherapeuticagents. H35 hepatoma cells untreated(NT ) or incubated with lovastatin (Lov )or YM-53601 for 24 h were exposed tothapsigargin (0.2 Amol/L), lonidamine(0.2 mmol/L), arsenic trioxide (5 Amol/L),doxorubicin (Doxo ; 1 Amol/L), bafilomycinA (Baf A ; 10 Amol/L) for 24 h. Celldeath was determined by trypan blueexclusion (n = 3). D, total cholesterol levelsof mitochondria from H35 and HepG2cells 24 h after treatment with lovastatin(2.5 Amol/L) or YM-53601 (1 Amol/L); n = 4;*, P < 0.05 versus untreated cells.Ctrl, control.

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Cholesterol impairs Bax-driven mitochondrial release ofapoptotic proteins and permeabilization in liposomes. Mito-chondrial release of prodeath factors by Bax or Bak can occur bytheir oligomerization and insertion into the mitochondrial outermembrane independent of MPT (22). Thus, we next examined theeffect of cholesterol on Bax-driven mitochondrial permeabilization.Whereas tBid-activated Bax (tBid/Bax) stimulated the release ofcytochrome c in control mitochondria, cholesterol-enrichedmitochondria were resistant to tBid/Bax-mediated cytochrome crelease (Fig. 5A). Similar findings were observed in the release ofSmac/Diablo by cholesterol enrichment (data not shown). Theincubation of cholesterol-enriched mitochondria with A2C, a fattyacid derivative that intercalates into the lipid bilayer resulting in itsfluidization (19), restored the ability of tBid/Bax to releasecytochrome c (Fig. 5A), establishing a cause-and-effect relationshipbetween mitochondrial membrane order and release of apoptoticproteins by Bax. Moreover, mitochondria from H35 cells wereinsensitive to tBid/Bax-induced release of cytochrome c that wasrestored by lovastatin treatment (Fig. 5A), which caused a

significant reduction in the mitochondrial cholesterol levels(Fig. 2D).

To further extend these obserations, we next examined the effectof cholesterol on the poration of large unilamellar vesicles (LUVs)by active Bax. LUVs with or without cholesterol were loaded withself-quenching concentrations of FD-70, and the release of LUV-entrapped FD-70 was monitored as an increase in the fluorescencesignal due to marker dilution in the external medium (24). Asshown, the release of vesicular content by tBid/Bax was impaired incholesterol-containing LUVs (Fig. 5B). In contrast, cholesterol wasrequired for the release of vesicular contents induced bytetanolysin, a cholesterol-dependent pore-forming toxin known toopen proteinaceous channels by inserting a transmembrane a-barrel in the bilayer (Fig. 5B ; ref. 39). Treatment of LUVs with MCDreversed the inhibitory effect of cholesterol upon Bax-drivenliposome permeabilization and disrupted the cholesterol-depen-dent channel-forming function of tetanolysin (Fig. 5B). Further-more, cholesterol inhibited the membrane-permeabilizing activityof tBid/Bax or Bax preoligomerized with octylglucoside (Oligo-Bax)

Figure 3. StAR suppression by siRNA increases the cytotoxicity induced by doxorubicin. A, representative immunoblot showing StAR protein abundance in H35and HepG2 cellular extracts and in homogenates from rat and human liver. B, StAR mRNA silencing. HepG2 cells were transfected with the StAR siRNA or the controlsiRNA (Ctrl siRNA ) as described in Materials and Methods. Cells were allowed to recover in regular culture medium for 48 h, and the levels of StAR protein werequantified by Western blot in cellular extracts (left ; n = 3) and mRNA levels by real-time RT-PCR (right ; n = 3). C, mitochondrial cholesterol levels from HepG2 and H35cells 48 h after StAR mRNA silencing analyzed by HPLC. D, 48 h after transfection with StAR or control siRNA, cells were exposed to doxorubicin (1 Amol/L),thapsigargin (0.2 Amol/L), lonidamine (Lnd ; 0.2 mmol/L), and arsenic trioxide (5 Amol/L), and cell death was analyzed 48 h later by trypan blue exclusion. At least100 cells in 4 different fields were counted and expressed as a percentage of total cells; n = 3; *, P < 0.05 versus control siRNA–transfected cells treated withdoxorubicin.





in a dose-dependent manner (Fig. 5B). Similar to the effect foundon Bax, and consistent with previous observations, cholesterol alsodecreased the permeabilizing activity of melittin, a widely studiedantimicrobial peptide thought to breach membrane permeabilitybarrier by forming lipid-containing toroidal pores instead of purelyproteinaceous channels (40). In contrast, similar to tetanolysin andconsistent with previous findings, cholesterol actually increasedthe release of vesicular contents induced by the channel-formingprotein S. aureaus a-toxin (Fig. 5B ; ref. 41). Antiapoptotic proteinssuch as Bcl-2 antagonize the release of mitochondrial prodeathfactors during apoptosis. To address whether cholesterol modu-lates Bcl-2 activity, LUVs with or without cholesterol were incu-bated with Bcl-2DC before tBid/Bax treatment. As seen, Bax-drivenmembrane permeabilization of LUVs was inhibited by Bcl-2DC in adose-dependent manner, despite the presence of membranecholesterol (Fig. 5C). Consistent with the findings in cholesterol-enriched rat liver mitochondria, the resistance to Bax-inducedpermeabilization of cholesterol-containing LUVs was reversed by

the fluidizing agent A2C (Fig. 5D). Finally, we examined whethercholesterol altered the membrane-inserting capacity of preoligo-merized Bax (Oligo-Bax) using lipid monolayers. Addition of oligo-Bax into cholesterol-containing or cholesterol-devoid lipid mono-layers at different initial surface pressures allowed us to determinecritical surface pressures values (k0) for oligo-Bax, which is ameasure of the membrane penetrability of the protein. As seen, k0

values for oligo-Bax in cholesterol-containing monolayers werenotably lower than in monolayers devoid of cholesterol (Fig. 5D).Together, these findings suggest that cholesterol-mediated rigidi-fication of the bilayer directly modulates Bax permeabilizingactivity, at least, in part, by reducing the capacity of Bax to insertinto the lipid matrix of the membrane.HMG-CoAR or SS inhibition potentiates chemotherapy in

tumor xenografts. We further evaluated whether cholesterolregulates cancer therapy in vivo using tumor xenografts. Nude micewere s.c. injected with human hepatoma HepG2 cells, and tumor-bearing animals were then subjected to lipid-lowering treatments.

Figure 4. Mitochondrial cholesterol modulation regulates the release of cytochrome c and Smac/Diablo by ROS and Ca2+. A, total cholesterol levels determinedby HPLC of untreated mitochondria or after cholesterol enrichment by a cholesterol-albumin complex (CHOL ) and Me-h-cyclodextrine treatment (MCD; top ). Middle,membrane order (SDPH ) determined from the fluorescence anisotropy monitored at 366 nm (emission, 440 nm) of mitochondria labeled with DPH, using polarizingfilters in both excitation and emission planes, and normalized as per milligram of mitochondrial protein.; n = 3; *, P < 0.05 versus untreated mitochondria; **, P < 0.05versus rat liver mitochondria. Bottom, correlation between mitochondrial cholesterol content and membrane order (SDPH). B, control and cholesterol-depleted (MCD)mitochondria from H35 and HepG2 cells were incubated with xanthine (0.1 mmol/L) plus xanthine oxidase (40 mU/mL; X-XO ), and the effect on the release ofcytochrome c was analyzed by Western blot in the supernatants at the indicated times. Rat liver mitochondria were exposed to X-XO, and the release of cytochrome cwas analyzed by Western blot in the supernatants. C, release of apoptogenic proteins by Ca2+ in control and cholesterol-enriched mitochondria from rat liver.Supernatants and pellets from mitochondria with or without CaCl2 (100 Amol/L) treatment for 1 h were used to analyze the levels of cytochrome c and Smac/DIABLO.D, representative immunoblots of cytochrome c and Smac/DIABLO in supernatants and pellets of control and Me-h-cyclodextrin–treated mitochondria from H35 cells,with or without CaCl2 (100 Amol/L) exposure for 30 min. B to D, the blots shown are representative of 3 to 4 independent experiments showing similar results.The mitochondrial hsp60 protein was visualized as a loading control.

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Atorvastatin was chosen for in vivo studies as it has been shown tobe more effective than other statins both in humans andexperimental animals (42, 43). We observed that both atorvastatinand YM-53601 administration significantly decreased the intra-tumor cholesterol levels (Fig. 6A). No differences in the pattern orin the amount of microvessel formation were observed in any ofthe experimental groups (Supplementary Fig. S8), suggesting thatcholesterol content plays a minor role in tumor vascularization inthis xenograft model. However, although atorvastatin notablyreduced the size of the tumors or the increase in a-feto protein

levels in serum (data not shown) after 2 weeks of administration,such effect was not observed with YM-53601 treatment (Fig. 6B),indicating that inhibition of tumor progression by statins may be inpart caused by isoprenoids down-regulation, in agreement withprevious studies (9, 10). Interestingly, however, mice treated withatorvastatin or the SS inhibitor displayed a greater reduction intumor growth after doxorubicin administration compared withuntreated mice (Fig. 6C). Furthermore, a higher number ofapoptotic cells assessed by TUNEL staining was observed intumors from atorvastatin or YM-53601 treated mice after

Figure 5. Cholesterol inhibits BAX-driven membrane permeabilization. A, representative immunoblots of cytochrome c in supernatants and pellets of mitochondriafrom control and cholesterol-enriched rat liver mitochondria (CHOL ) with or without A2C incubation (125 nmol/mg protein) after 45 min of tBid+Bax (20 nmol/L) treatment(left). Right, representative immunoblots of cytochrome c in supernatants and pellets of mitochondria from H35 cells treated or not with lovastatin (2.5 Amol/L)followed by 45 min of tBid+Bax (20 nmol/L) treatment. B, representative kinetics of BAX + tBID- (50 nmol/L) and tetanolysin-induced (10 nmol/L) FD70 release fromLUV composed of phosphatidylcholine (PC)/cardiolipin (CL) 90/10 (mol/mol; �CHOL) or PC/CL/cholesterol 45/10/45 (mol/mol; +CHOL ; left). Middle, BAX+tBID-(BAX ; 50 nmol/L) and tetanolysin-induced (TET ; 10 nmol/L) FD70 release from LUV, in the presence or absence of 2 mmol/L methyl-h-cyclodextrin MCD (n = 2).Right, vesicular FD-70 release induced by BAX+tBID (20 nmol/L), BAX oligomerized (oligo-BAX; 50 nmol/L), melittin (200 nmol/L), S. aureaus a-toxin (a toxin;20 nmol/L), and tetanolysin (5 nmol/L) in LUV composed of PC/CL 90/10 (mol/mol) in which PC was substituted by increasing amounts of cholesterol (n = 3–6).C, FD-70 release in LUV composed as in B and exposed to indicated amounts of Bcl-2 for 5 min before treatment with BAX+tBID (50 nmol/L). The data are normalizedas a percentage of the release produced by BAX+tBID in the absence of Bcl-2 (n = 2). D, dose-dependent effect of A2C on FD70 release from LUV composedof PC/CL/CHOL 45/10/45 (mol/mol) in which PC was substituted by increasing amounts of A2C and then exposed to Bax and tBid (50 nmol/L each; left). Columns,mean two to four independent measurements; bars, SD. Right, cholesterol reduces the ability of oligomerized Bax by octylglucoside to penetrate into lipidmonolayers at different surface pressures. Lipid composition was PC/CL 80/20 (�CHOL) and PC/CL/CHOL 40/20/40 (+CHOL). Critical surface pressures valueswere 34.6 mN/m (�CHOL) and 30.2 mN/m (+CHOL). Oligomeric Bax concentration was 400 nmol/L.





doxorubicin administration compared with doxorubicin alone(Fig. 6D), indicating an increased susceptibility of HCC to thechemotherapeutic agent. Together, these data highlight thepotential relevance of the increased mitochondrial cholesterol inmodulating the response of HC to chemotherapy in vivo .

Discussion

Since its description decades ago in heterotopic Morrishepatoma xenografts in Buffalo rats, the mitochondrial enrichmentin cholesterol has been viewed mainly as a key factor underlying, inpart, the mitochondrial dysfunction characteristic of cancer cells(7). In this study, however, we investigated the role of cholesterolaccumulation in mitochondria from HCC in response to chemo-therapy in vitro and in vivo and the mechanisms involved. First, weconfirmed that established human and rat HCC exhibit increasedmitochondrial cholesterol levels with respect to nontumor mito-chondria of human and rat liver, as reported in solid hepatomamitochondria compared with host liver (12–15), which paralleledthose found in mitochondria from primary tumors of patients withHC. Close to 70% of the cholesterol found in mitochondria fromHCC was unsterified, which, in contrast to esterified cholesterol, isknown to regulate membrane dynamics and order (1, 2, 19, 27, 28).

Moreover, the up-regulation of mitochondrial cholesterol contentin HCC correlates with increased expression of SREBP-2 andHMG-CoAR, thus validating the cholesterol deregulation of cancercells (7–9) and their use to examine the susceptibility tochemotherapy.

Our data uncover the resistance of HCC to chemotherapy-induced cell death that was reversed when cholesterol levels werereduced by inhibition of HMG-CoAR or SS. Although this strategydepletes cholesterol in different types of membranes other thanmitochondria such as in specific domains of the plasmamembrane, which has been described to modulate cancer therapyby regulating survival pathways such as Akt (5, 6), we provideevidence supporting a specific role for the mitochondrialcholesterol in modulating the susceptibility of HCC to chemother-apy. First, we show that the sensitivity of HepG2 and H35 cells toarsenic trioxide and lonidamine is potentiated by cholesteroldepletion by HMG-CoAR or SS inhibition. Arsenic trioxide andlonidamine both induced MPT-mediated apoptosis by targeting theadenine nucleotide translocator (ANT)1 isoform (30, 31). Four ANTisoforms have been described, which are encoded by closely relatedgenes that belong to the mitochondrial carrier family, and ANT is amajor component of the MPT that mediate MMP and cell death(44). Mitochondria from murine cells lacking ANT 1 and ANT2 can

Figure 6. Reduction of cholesterol synthesis potentiates doxorubicin therapy in an in vivo murine model. HepG2 cells (2.5 � 106 cells per animal) were implanteds.c. in the flanks of athymic mice as described in Materials and Methods. When tumors averaged 50 mm3 in size (2–3 wk), mice were randomly divided into theexperimental groups and treated by a daily p.o. gavage with atorvastatin (10 mg/kg body weight) or YM-53601 (15 mg/kg body weight) for 2 wk. A, cholesterollevels from control (vehicle treated), atorvastatin, and YM-53601–treated tumors. (n = 5). *, P < 0.05 versus control. B, tumor growth measured periodically at theindicated days over the treatment. The volume was calculated as length � width2 � 0.5. The experiment was performed twice with similar results. *, P < 0.05 versusvehicle-treated tumor. C, after 2 wk of treatment, some animals received an i.p. injection of doxorubicin (10 mg/kg). Fold increase of tumor growth was evaluatedin each experimental group (n = 5) by calculating the changes in tumor size observed after administration of the chemotherapy agent. The experiment was performedtwice with similar results. *, P < 0.05 versus doxorubicin-treated control mice. D, apoptosis induced by the chemotherapy agent in tumors from animals treatedeither with statins or SS inhibitor was analyzed by TUNEL-positive staining areas. Representative images of three samples per group performed showing similar results.

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still undergo Ca2+-induced swelling and MPT, although at a higherthreshold (45), which have been viewed as evidence against arole for ANT in MPT and hence in MMP. However, the abilityof ANT1/ANT2-deficient cells to undergo MMP might be due tothe functional compensation by a novel ANT isoform identifiedrecently (46) or by other mitochondrial carriers able to form poresin the inner membrane, such as the ornithine/citrulline trans-porters or the phosphate carrier (47).

Furthermore, additional evidence for a role of mitochondrialcholesterol in chemotherapy susceptibility derives from theoutcome observed by StAR silencing. StAR is a polypeptideresponsible for the intramitochondrial transport of cholesterol(11, 16) and both HepG2 and H35 cells overexpress StAR levels.StAR silencing by siRNA sensitizes these cell lines to chemo-therapy-induced cell death. Consistent with its role in themitochondrial trafficking of cholesterol, the sensitization tochemotherapy by StAR down-regulation could reflect impairedcholesterol transport from the outer to the inner mitochondrialmembranes. However, our data indicate that StAR depletion bysiRNA resulted in the net decrease of mitochondrial cholesterollevels, implying lower delivery of cholesterol to mitochondriafrom extramitochondrial sources by other proteins including(StAR)-related lipid transfer (StART) family members. Althoughthe molecular mechanisms of StAR are poorly understood, recentfindings have shown that StAR interacts with specific outermitochondrial membrane proteins such as VDAC1 and thephosphate carrier protein (48). Whether StAR works in concertwith other StART members via interaction with specific proteinsof the outer mitochondrial membrane to deliver cholesterol tomitochondria remains to be investigated. Thus, collectively, thesedata strongly suggest that the enrichment of mitochondria incholesterol plays a role in the resistance of chemotherapy actingvia mitochondria.

To explore the mechanism of the mitochondrial cholesterol–mediated resistance to chemotherapy, we focused on themembrane properties of isolated mitochondria from HCC. Weobserved that mitochondria from HepG2 and H35 cells exhibithigher order which is reversed by cholesterol extraction withMCD or fluidization by A2C, translating in increased MMP andrelease of cytochrome c and Smac/Diablo in response to Ca2+,superoxide anion, and active Bax. An important piece of evidencesupporting the specificity of cholesterol in the regulation of MMPis the fact that the in vitro enrichment of rat liver mitochondriain cholesterol to about the same levels seen in H35 cellsreproduces the resistance to MMP and release of intermembraneapoptotic proteins induced by active Bax. Consistent with theseobservations, cholesterol-containing LUVs are also resistant to theporation induced by Bax, and cholesterol dose-dependentlyinhibited the vesicular release induced by melittin, which hasbeen proposed to permeabilize membranes by forming toroidallipidic pores (40). Because positive monolayer curvature stresscontributes to MMP by Bax (24), it is conceivable that cholesterolmay stabilize mitochondrial membranes against Bax permeabiliz-ing action through induction of negative curvature, consistentwith the accumulation of cholesterol in high-curvature regions ofmembranes (49). Remarkably, cholesterol decreased the capacityof Bax to penetrate into the membrane, which is considered to bea critical step in Bax activation. Thus, through modulation ofmembrane order and curvature stress, cholesterol may regulateBax activity and the formation of lipidic pores in mitochondrialmembranes and, hence, cell death susceptibility.

We also observed that tumor growth and chemotherapysusceptibility in heterotopic murine tumor xenografts weremodulated by HMG-CoAR or SS inhibition, with both strategiesdecreasing tumor mitochondrial cholesterol levels. The generationof farnesyl diphosphate from mevalonate branches into isopre-noids and squalene, which is then committed for cholesterolsynthesis (Supplementary Fig. S4). Isoprenoids are known tomodify the function of proteins through posttranslational mod-ifications such as the Rho family of small GTPases that coordinatesmany aspects of cell motility through the reorganization of actincytoskeleton and changes in gene transcription (9, 10). Theisoprenylation is essential for Rho-mediated invasion of varioustumors, including melanoma, pancreatic, and breast cancer cells(50). In the in vivo xenograft model, we observed that HMG-CoARbut not SS reduced tumor growth, suggesting that inhibition oftumor progression by statins may be in part caused by isoprenoidsdown-regulation. However, the susceptibility of HC xenograftsto chemotherapy are observed upon statins or SS inhibition,supporting the relevance of cholesterol rather than isoprenoidsgeneration in the resistance of HCC to chemotherapy. Thus, ourfindings regarding the role of SS inhibition on chemotherapy alongwith recent reports in different types of cancer cells such as humanprostate carcinoma cell lines (51) highlight the relevance ofcholesterol modulation in cancer therapy.

Interestingly in line with our data, recent findings in liposomesconfirmed that mitochondrial Bax activation is inhibited bycholesterol (52). Moreover, mitochondria from HeLa cells treatedwith U18666A, which caused mitochondrial cholesterol up-regulation, showed a delay in the release of Smac/Diablo andcytochrome c , as well as in Bax oligomerization and partialprotection against stress-induced apoptosis (52).

In summary, although enhanced cholesterol levels in cellmembranes including mitochondria from cancer cells have beenlong known, we show here for the first time the relevance of thecholesterol-mediated regulation of mitochondrial membranedynamics in the response of HCC to mitochondrial-targetingchemotherapy in vitro and in vivo. Moreover, the potentiation ofHC chemotherapy by SS inhibition, which reduces cholesterollevels including in mitochondria, without perturbing isoprenoidmetabolism, validates the specificity of cholesterol in chemother-apy resistance, and revitalizes the potential benefit of cholesteroldown-regulation in cancer therapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Received 11/8/2007; revised 3/14/2008; accepted 4/16/2008.Grant support: Research Center for Liver and Pancreatic Diseases Grant P50 AA

11999 funded by the US National Institute on Alcohol Abuse and Alcoholism; PlanNacional de I+D Grants SAF2005-03923, SAF2005-03943, SAF2006-06780, BFU2005-06095, and FIS06/0395; the Centro de Investigacion Biomedica en Red deEnfermedades Hepaticas y Digestivas supported by the Instituto de Salud Carlos III;the Ramon y Cajal Research Program (Ministry of Education and Science; A. Colelland A. Morales); and a predoctoral fellowship from the Basque Government(O. Terrones).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Susana Nunez for the technical assistance and Dr. Bataller for thedonation of human liver samples; Virginia Villar from Clinic and Center for AppliedMedical Research, Pamplona, for her assistance in providing human HC samples; andthe Servicio Cientifico-Tecnico of IDIBAPS for electron microscopy, confocal imaging,and flow cytometry analysis.





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2008;68:5246-5256. Cancer Res Joan Montero, Albert Morales, Laura Llacuna, et al. Resistance in Hepatocellular CarcinomaMitochondrial Cholesterol Contributes to Chemotherapy

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